私たちは、彼女の優れた技術支援のためのKristaデンOudenに感謝します。この手法は、財政的にオランダの腎臓財団、助成C06.2174によってサポートされていました。 MCVは、科学研究のためのオランダ組織（NWO）ビディ·助成金016.096.359によってサポートされています。

全ての実験は、ユトレヒト実験動物委員会の動物実験の倫理的なガイドラインに基づいて実行されます。プロトコルは、著者の機関の動物のケアと使用委員会の指導と承認の下で行われる。 CKDは、8週齢の雄の近交系Lewisラット（チャールズリバー、ズルツフェルト、ドイツ）に誘導される。ラットには、光、温度および湿度制御された環境での標準条件下で飼育されています。 1。手術の準備<ol><li>手術器具を殺菌する： <ul><li> 1学生組織鉗子1-2歯12センチメートル</li><li> 1学生の標準パターンの鉗子</li><li> 2 Semken鉗子</li><li> 1マヨはさみ</li><li> 1生徒アイリスはさみ</li><li>ハサミで1オルセン-ヘーガル針ホルダー</li><li>毛布</li></ul></li><li>インベントリリストをチェックします。 <ul><li>温暖化パッドとランプでテーブルを操作する</li><li> O <sub&gt;の2、イソフルラン</li><li>組織</li><li>シェーバー</li><li>秤</li><li>無菌操作セット</li><li>滅菌ガーゼの5×5と10×10 </li><li> 70％アルコール</li><li> 0.9％NaCl溶液</li><li> 、1mlの注射器</li><li>針（25G） </li><li>ゲルフォームパッド：spongostan </li><li> VICRYL 4.0および5.0 </li><li>ブプレノルフィン0.03 mg / kgを（生理食塩で1:10に希釈） </li><li>手術後のラットクリーンケージ</li></ul></li></ol> 2。右サイドUninephrectomy <ol><li> 70％アルコールでテーブルを消毒。 </li><li>場所は、インダクションボックスにラットおよび4％イソフルランとO 2の1リットル流れに麻酔を誘導する。注：使用されるラット系統に応じて、事前と周術期鎮痛が処方されて、これは地元の獣医師と協議する必要があります。 </li><li>テーブルと場所ノーズコーンににラットを移す。ラットおよびノー​​ズコーンは、ラットの体温を維持するために、加熱パッド上に配置される。ラットIsが2％イソフルラン1リットルとO 2との混合物で換気。 </li><li>ラットの側面を剃るとアルコールで消毒。洗って消毒の手を、無菌手術用手袋を着用してください。ヘッドキャップとマスクを着用してください。 1を作る - 解剖鉗子と鈍いハサミを使って肋骨に平行1.5センチメートル切開。背中の筋肉の鈍的切開によって腎臓を公開します。 </li><li>腎臓の下極が表示され、慎重に腎周囲脂肪組織を把持する小さな鈍鉗子を使用しています。優しく鉗子と腎周囲脂肪を引いて腎臓を外部化。この手順の実行中に副腎を妨げないように注意してください。副腎は容易に脂肪組織における中間部位に鉗子を配置することにより除去し、 図2に示すように穏やかに副腎と腎臓との間を上方に移動させることができる。 </li><li>ガーゼや脂肪と結合組織の周囲の明確に優しく腎臓を置きます。腎動脈と静脈を特定し、合字（5.0を配置船の周りにシングルノットとVICRYL）が、結び目を縛るません。 </li><li>腎臓と結び目の間にスペースを作成するために、およそ0.5㎝大動脈に向かって血管に沿って慎重に緩い結び目を移動します。これは、切削後に結紮糸抜けを防ぐために行われる。 </li><li> 2ダブルノットで結び目を作る。灌流が停止された場合、腎臓の色はすぐに茶色に変わります。合字の両端を切断しないでください。 </li><li>腎臓に近い腎血管をカットし、腎臓を削除します。優しく腎血管の出血をチェックするリガチャーの長い両端を引っ張る。リガチャーは、容器上の場所に滞在する必要があります。リガチャーの長い端をカットします。注意、残存腎血管がすぐに腹腔内に収納されます。 </li><li>しみ摘出腎臓が乾燥し、それを重量を量る。 </li><li> VICRYL 5.0で縫合を実行すると骨格筋切開を閉じます。 0.10ミリリットル（0.03 mg / kg）を後肢でブプレノルフィンIMを注入。クローズ皮内縫合とVICRYL 4.0と肌。これは傷口を開くラットを防ぐことができます。注：ルイスラットの呼吸抑制のために、我々はばかりイソフルランをオフにする前に、術後鎮痛を与える。 </li><li>食べ物や水へのアクセスが簡単きれい孤独なケージにラットを収容。温暖化パッドO / Nでケージ半分を置き、慎重にラット翌日を確認してください。創傷は、通常、手術後6〜12時間である、閉じられ、ラットがアクティブであるときに、ラットをグループハウジングに戻って配置することができる。 </li></ol> 3。左側小計腎摘出<ol><li> 2.1から2.5に説明されているように7日後、同製剤は、右側用と左側に作られています。手術前、右腎臓の重量の約3分の2に相当除去する必要がある腎組織の量が算出される。手術前にゲルフォームの小片（Spongostan、ジョンソン＆ジョンソン、ニュージャージー州）を準備。 </lI&gt; <li>左腎上極の周りに鋭いハサミを置き、1ストローク（はさみ位置については、 図3を参照）、上部ポールを切除。ゲル泡の部分ですぐにカバーし、滅菌ガーゼと軽度の圧力をかける。 </li><li> 3.3のように、腎臓の下極と繰り返します。 </li><li>皮膚上の凝固を防止するために腎臓を持ち上げます。出血が止まるまで、滅菌ガーゼでゲルフォームパッドの両方で軽度の圧力を維持する。出血が持続する場合は、別のフォームパッドを追加します。 </li><li>腹部内部の付着ゲルフォームパッドと残存腎臓を置きます。筋肉と皮膚を閉じて、2.9から2.12に従ってください。 </li></ol> 4。偽手術<ol><li>偽手術コントロールは、腎臓を露出させるために同じ手順を受ける。代わりに腎臓を摘出または一週間後極の切断、腎臓両方が副腎を乱さないように注意しながら、1週間間隔でカプセル化が解除されています。創傷閉鎖と術後のケアは、idである2.11と2.12にentical。 </li></ol>

<ol>
	<li>AS, G. o., Chertow, G. M., Fan, D., McCulloch, C. E., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+kidney+disease+and+the+risks+of+death,+cardiovascular+events,+and+hospitalization.">Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization.</a> N. Engl. J. Med. 351 (13), 1296-1305 (2004).</li><li>Fleck, C., Appenroth, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+5/6+nephrectomy+(5/6NX)+for+the+induction+of+interstitial+renal+fibrosis+in+rats--influence+of+sex,+strain,+and+surgical+procedure.">Suitability of 5/6 nephrectomy (5/6NX) for the induction of interstitial renal fibrosis in rats--influence of sex, strain, and surgical procedure.</a> Exp. Toxicol. Pathol. 57 (3), 195-205 (2006).</li><li>Griffin, K. A., Picken, M. M., Churchill, M., Churchill, P., Bidani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+correlates+of+glomerulosclerosis+after+renal+mass+reduction+in+the+rat.">Functional and structural correlates of glomerulosclerosis after renal mass reduction in the rat.</a> J. Am. Soc. Nephrol. 11 (3), 497-506 (2000).</li><li>Kang, D. H., Kanellis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+microvascular+endothelium+in+progressive+renal+disease.">Role of the microvascular endothelium in progressive renal disease.</a> J. Am. Soc. Nephrol. 13 (3), 806-816 (2002).</li><li>Baylis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+derangements+and+hypertension+in+kidney+disease.">Nitric oxide synthase derangements and hypertension in kidney disease.</a> Curr. Opin. Nephrol. Hypertens. 21 (1), 1-6 (2012).</li><li>Bongartz, L. G., Braam, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+nitric+oxide+reduction+induces+permanent+cardiac+systolic+dysfunction+and+worsens+kidney+damage+in+rats+with+chronic+kidney+disease.">Transient nitric oxide reduction induces permanent cardiac systolic dysfunction and worsens kidney damage in rats with chronic kidney disease.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 298 (3), 815-823 (2010).</li><li>Fujihara, C. K., De N, G., Zatz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nitric+oxide+synthase+inhibition+aggravates+glomerular+injury+in+rats+with+subtotal+nephrectomy.">Chronic nitric oxide synthase inhibition aggravates glomerular injury in rats with subtotal nephrectomy.</a> J. Am. Soc. Nephrol. 5 (7), 1498-1507 (1995).</li><li>Fujihara, C. K., Sena, C. R., Malheiros, D. M., Mattar, A. L., Zatz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+nitric+oxide+inhibition+induces+progressive+nephropathy+after+regression+of+initial+renal+injury.">Short-term nitric oxide inhibition induces progressive nephropathy after regression of initial renal injury.</a> Am. J. Physiol. Renal Physiol. 290 (3), F632-F640 (2006).</li><li>Dikow, R., Kihm, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+infarct+size+in+uremic+rats:+reduced+ischemia+tolerance?.">Increased infarct size in uremic rats: reduced ischemia tolerance?.</a> J. Am. Soc. Nephrol. 15 (6), 1530-1536 (2004).</li><li>Elrashidy, R. A., Asker, M. E., Mohamed, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pioglitazone+attenuates+cardiac+fibrosis+and+hypertrophy+in+a+rat+model+of+diabetic+nephropathy.">Pioglitazone attenuates cardiac fibrosis and hypertrophy in a rat model of diabetic nephropathy.</a> J. Cardiovasc. Pharmacol. Ther. 17 (3), 324-333 (2012).</li><li>Haylor, J., Schroeder, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+gadolinium+following+use+of+MR+contrast+agents+in+a+rat+model+of+nephrogenic+systemic+fibrosis.">Skin gadolinium following use of MR contrast agents in a rat model of nephrogenic systemic fibrosis.</a> Radiology. 263 (1), 107-116 (2012).</li><li>Moriguchi, Y., Yogo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+hypertrophy+is+associated+with+inflammation+in+sodium+loaded+subtotal+nephrectomized+rats.">Left ventricular hypertrophy is associated with inflammation in sodium loaded subtotal nephrectomized rats.</a> Biomed. Res. 32 (2), 83-90 (2011).</li><li>van Koppen, A., Joles, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthy+bone+marrow+cells+reduce+progression+of+kidney+failure+better+than+CKD+bone+marrow+cells+in+rats+with+established+chronic+kidney+disease.">Healthy bone marrow cells reduce progression of kidney failure better than CKD bone marrow cells in rats with established chronic kidney disease.</a> Cell Transplant. , (2012).</li><li>Baylis, C., Corman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+kidney:+insights+from+experimental+studies.">The aging kidney: insights from experimental studies.</a> J. Am. Soc. Nephrol. 9 (4), 699-709 (1998).</li><li>Szabo, A. J., Muller, V., Chen, G. F., Samsell, L. J., Erdely, A., Baylis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number+determines+susceptibility+to+renal+mass+reduction-induced+CKD+in+Lewis+and+Fisher+344+rats:+implications+for+development+of+experimentally+induced+chronic+allograft+nephropathy.">Nephron number determines susceptibility to renal mass reduction-induced CKD in Lewis and Fisher 344 rats: implications for development of experimentally induced chronic allograft nephropathy.</a> Nephrol. Dial Transplant. 23 (8), 2492-2495 (2008).</li><li>Darling, I. M., Morris, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+"true"+creatinine+clearance+in+rats+reveals+extensive+renal+secretion.">Evaluation of "true" creatinine clearance in rats reveals extensive renal secretion.</a> Pharm. Res. 8 (10), 1318-1322 (1991).</li><li>Levey, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+renal+function+in+chronic+renal+disease.">Measurement of renal function in chronic renal disease.</a> Kidney Int. 38 (1), 167-184 (1990).</li><li>Myers, G. L., Miller, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+improving+serum+creatinine+measurement:+a+report+from+the+Laboratory+Working+Group+of+the+National+Kidney+Disease+Education+Program.">Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program.</a> Clin. Chem. 52 (1), 5-18 (2006).</li><li>Hostetter, T. H., Meyer, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+clearance+methods+for+measurement+of+glomerular+filtration+and+tubular+reabsorption.">The development of clearance methods for measurement of glomerular filtration and tubular reabsorption.</a> Am. J. Physiol. Renal Physiol. 287 (5), F868-F870 (2004).</li><li>Koeners, M. P., Racasan, S., Koomans, H. A., Joles, J. A., Braam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide,+superoxide+and+renal+blood+flow+autoregulation+in+SHR+after+perinatal+L-arginine+and+antioxidants.">Nitric oxide, superoxide and renal blood flow autoregulation in SHR after perinatal L-arginine and antioxidants.</a> Acta. Physiol. (Oxf). 190 (4), 329-338 (2007).</li><li>van den Brandt, J., Wang, D., Kwon, S. H., Heinkelein, M., Reichardt, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirally+generated+eGFP-transgenic+rats+allow+efficient+cell+tracking+in+vivo.">Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo.</a> Genesis. 39 (2), 94-99 (2004).</li><li>Kreutz, R., Kovacevic, L., Schulz, A., Rothermund, L., Ketteler, M., Paul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+NaCl+diet+on+spontaneous+hypertension+in+a+genetic+rat+model+with+reduced+nephron+number.">Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number.</a> J. Hypertens. 18 (6), 777-782 (2000).</li><li>Liu, Z. C., Chow, K. M., Chang, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+protocols+of+uremic+rat+model:+partial+nephrectomy+and.">Evaluation of two protocols of uremic rat model: partial nephrectomy and.</a> 25 (6), 935-943 (2003).</li><li>Griffin, K. A., Picken, M., Bidani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+renal+mass+reduction+is+a+critical+modulator+of+subsequent+hypertension+and+glomerular+injury.">Method of renal mass reduction is a critical modulator of subsequent hypertension and glomerular injury.</a> J. Am. Soc. Nephrol. 4 (12), 2023-2031 (1994).</li><li>Meyer, F., Ioshii, S. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+partial+nephrectomy+in+rats.">Laparoscopic partial nephrectomy in rats.</a> Acta. Cir. Bras. 22 (2), 152-156 (2007).</li></ol>

著者らは、開示することは何もありません。

高塩食と一時的NOS阻害と組み合わせルイスラットにおける腎腫瘤の5/6thの外科的除去は、人間に似ているCKD CKDのモデルにつながり、CKDにおける治療的介入の因果メカニズムと有効性の研究を可能にします。 5/6thの腎摘出モデルでは、CKDのためのよく知られており、広範囲に記述されたモデルです。しかし、単に腎腫瘤の5/6thを削除すると、すべてのラット系統における即...

その進歩的な性質、その後の末期腎疾患、および関連する心血管系の罹患率および死亡率のため、CKDは、成長公衆衛生上の問題1です。 CKDの進行を遅くするので、主要な健康上の優先順位です。 CKDの恒常性の複合撹乱することを特徴とするので、統合的な動物モデルは、CKDの発症および進行を研究するために必要である。腎臓は、互いに相互作用の異なる細胞型の広い範囲から構成される。この複雑さは、 インビトロで模倣することができない。 CKDの新たな治療介入を研究するために、我々は人間のCKD 2,3のいくつかの側面に似ている、5/6th腎アブレーションモデル、進行性腎疾患のよく知られた実験モデルを使用しています。腎質量の総減少はプログレッシブ糸球体と尿細管間質傷害、残留ネフロンの損失と全身および糸球体高血圧の開発を引き起こす。これは、プログララマブルに関連付けられてい様格腎内毛細血管損失4、炎症や糸球体硬化症。の危険因子は、常に内皮機能5への影響をCKD。我々は、CKDの開発に比較的抵抗であるため、我々は一酸化窒素（NO）枯渇6、図7、図8及び高塩食9腎腫瘤の5/6thの除去を組み合わせたラット系統（ルイス）を使用した。到着と馴化後、動物は、2日後にL-NNAの継続と右サイドuninephrectomy（UNX）が続いて4週間の期間のために飲料水（20 mg / Lで）に補充NO合成酵素阻害剤（L-NNA）は、受信しません。一週間後、腎質量の3分の2の小計腎摘出術（SNX） すなわち除去は左側に行われます。 SNX後、動物を4週間の期間の飲料水に20 mg / LでLNNA続いて再び2日間回復させる。グランド飼料（タイムライン図1参照）に補充高塩食（6％）は、実験を通して継続される。 T彼は右側にUNXを実行する理由および左側SNXは、腎臓が身体の外部に露出されたとき、腎血管がそれが簡単すぎて血管を伸ばすことなく、腎臓にアクセスできるようになり左側に長いということである。文献で ​​は、モデルは、一週間後10,11,12右腎のUNX続いて、左腎の極を最初に除去されるで説明されています。我々の手では、このモデルは、腎不全のはるかに急速な発展を示したが、さらに腎機能の損失をはるかに大きい変化を示す。腎不全の進行は、プラズマ尿素、収縮期血圧およびタンパクを測定することによって、経時的に追跡される。 SNX後6週間、腎不全は、開発した糸球体濾過率（69％）および有効腎血漿流量の顕著な減少（62％）13高血圧（収縮期血圧&gt; 150 mmHgで）ことを特徴とする、タンパク尿（&gt; 50 mg/24時間）と軽度の尿毒症（&gt; 10 mm）である。組織学的特徴はtubul含める炎症、管状の萎縮と線維症とレムナント人口（&lt;10％）以内に健康球体の大規模な削減につながる巣状糸球体硬化によって反射されたO-間質損傷。フォローアップ12週後までSNXは、治療介入の評価のための機会の窓を提供し、CKDのさらなる進行を示しています。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>L-NNA</td>
			<td>Sigma-aldrich</td>
			<td>N5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spongostan dental: gel foam pads 1x1x1 cm</td>
			<td>Johnson&amp;Johnson</td>
			<td>Ms0005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl FS-2S naald 4/0 V392H p/36</td>
			<td>Ethicon</td>
			<td>V303H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl RB-1+ naald 5/0 V303H p/36</td>
			<td>Ethicon</td>
			<td>V392H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (0.3 mg/ml)</td>
			<td>Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals</td>
			<td>unknown</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Student Tissue Forceps - 1x2 Teeth 12 cm</td>
			<td>Fine Science Tools (FST)</td>
			<td>91121-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forceps</td>
			<td>FST</td>
			<td>91100-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>FST</td>
			<td>14010-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2X Semken Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Iris Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>FST</td>
			<td>12002-14</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the lewis rat

慢性腎臓病（CKD）は、グローバルな問題である。 CKDの進行を遅くすることは主要な健康上の優先順位です。 CKDの恒常性の複合撹乱することを特徴とするので、統合的な動物モデルは、CKDの発症および進行を研究するために必要である。 CKDにおけるCKDおよび新規治療的介入の開発を研究するために、我々は人間のCKDのいくつかの側面に似ている、5/6th腎アブレーションモデル、進行性腎疾患のよく知られた実験モデルを使用しています。腎質量の総減少はプログレッシブ糸球体と尿細管間質傷害、残留ネフロンの損失と全身および糸球体高血圧の開発を引き起こす。また、進行性の腎内毛細血管損失、炎症や糸球体硬化症に関連付けられています。の危険因子は、常に内皮機能への影響をCKD。これを模倣するために、私たちは、一酸化窒素（NO）の枯渇と高塩分食を腎腫瘤の5/6thの除去を兼ね備えています。到着と順応した後、動物は、再右サイドuninephrectomy続い4週間の期間のために飲料水（20 mg / Lで）に補充NO合成酵素阻害剤（NG-ニトロ-L-アルギニン）（L-NNA）は、ceiveん。一週間後、小計腎（SNX）は左側に行われます。 SNX後、動物を4週間のさらなる期間の飲料水（20 mg / Lで）でLNNA、続いて二日間回復させる。グランド飼料（タイムライン図1参照）に補充高塩食（6％）は、実験を通して継続される。腎不全の進行は、プラズマ尿素、収縮期血圧およびタンパクを測定することによって、経時的に追跡される。 SNX後6週間で、腎不全が開発しました。腎機能は、 &#39;ゴールドスタンダード&#39;イヌリンおよびパラ - アミノ馬尿酸（PAH）クリアランス技術を用いて測定される。 CKDのこのモデルは、糸球体濾過率（GFR）の減少と有効腎血漿流量（エルプ）、高血圧（収縮期血圧&gt; 150 mmHgで）、タンパク尿（&gt; 50 mg/24時間によって特徴付けられる）と軽度の尿毒症（&gt; 10 mm）である。組織学的特徴は、炎症、管状の萎縮と線維症とレムナント人口（&lt;10％）以内に健康球体の大規模な削減につながる巣状糸球体硬化によって反射された尿細管間質損傷も含まれます。 SNXはCKDのさらなる進行を示した後、12週までフォローアップ。

小計腎摘出後、全腎質量の約6分の1が残っている。 図4は 、前の2つの実験の平均と標準偏差が右腎の取り外された部品の重量を示しています。削除する必要がある重量は5/6th除去足らずで右腎常に結果の重量に基づいて計算されたことを示す、一つはUNX週間後に、左腎の肥大が起こるということを覚えておいてください。しかしながら、手術中に左腎の量を測定することは不可能...

Watch this Scientific Journal Video about 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat at JoVE.com

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

外科的腎腫瘤の5/6thを除去することによりルイスラットの慢性腎臓病（CKD）を確立するために二段階法が記載されている。外科手術、NOS阻害および高塩食の組み合わせは、因果メカニズムと新たな治療介入の開発の研究を可能にし、人間のCKDに似たモデルにつながる。

高塩ダイエットとルイスラットにおける慢性腎臓病を誘発するために一酸化窒素合成酵素阻害と組み合わせた5/6thの腎摘出

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

56th-nephrectomy-combination-with-high-salt-diet-nitric-oxide

Research

JoVE Journal

Medicine

23.4K ビュー.  University Medical Center Utrecht. 外科的腎腫瘤の5/6thを除去することによりルイスラットの慢性腎臓病（CKD）を確立するために二段階法が記載されている。外科手術、NOS阻害および高塩食の組み合わせは、因果メカニズムと新たな治療介入の開発の研究を可能にし、人間のCKDに似たモデルにつながる。

ビデオ: 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

一酸化窒素の生物活性をアッセイするための分析手法

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

医学

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

心房細動に対する左心耳閉鎖との組み合わせでカテーテルアブレーション

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

多発性嚢胞腎疾患で、ヒトとマウスの染色体異常を研究するためにスペクトル核型分析

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

ラットにおける慢性栄養注入のモデル

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

慢性腎臓病患者における血管機能の評価

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

ラット網膜へのエルバックス40Wを通じて徐放性ドラッグデリバリー：慢性症状の治療のための含意

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

マウスにおける安定な高レベルの骨髄キメラ現象を生成するための骨髄抑制剤としてブスルファン

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

ラット腎臓への生物製剤の血管内デリバリー

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

糸球体疾患のマウス ・ モデルにおける腎機能の評価

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

VASP のリン酸化を測定することによって血液中の一酸化窒素の血小板ベースの検出

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...